Project Summary/Abstract Blood is a dynamic tissue that continuously regenerates, with replacement of approximately 1011 cells per day. This process of hematopoiesis is driven by hematopoietic stem cells (HSCs) that have the capacity to make all blood lineages upon transplantation. Notably, much of our knowledge of the hematopoietic system has come from bulk transplantation or in vitro colony formation assays. The advent of novel lineage tracing methods and single-cell technologies, however, has led to a dramatic shift in our view of HSCs. Indeed, as a population, they have been shown to be remarkably heterogeneous, both transcriptionally and in terms of lineage output. Still unresolved is how HSCs decide upon particular fates during development, and how these fate decisions are altered during disease or aging. As we continue to revise our view of HSCs, questions about the ontogeny of the hematopoietic system--from where stem and progenitor cells arise, how they mature, and how they ultimately establish the nascent blood system--are of significant biological and clinical interest. How many unique clones are active at a given time, and do they act independently or as a consortium in generating downstream progeny? To trace blood development in situ and at a clonal level, our lab has developed a novel genetic mouse model that utilizes random transposon integration to barcode cells. Upon induction, each cell is marked by a stable, genetic tag that is inherited by its progeny; at a later time, these tags can then be used to infer lineage relationships and map developmental hierarchies. Due to the large diversity of possible barcodes, this tool will allow me to lineage trace complex populations such as embryonic HSCs and, for the first time, clonally examine their behavior in a native setting. Here, I propose to utilize this tool to understand how blood stem and progenitor cells establish the hematopoietic system and to generate a map of the hematopoietic hierarchy during development. First, I will assess the number and contribution of HSCs that are specified during embryogenesis. Second, I will characterize the origins and clonal dynamics of short-term HSCs and multipotent progenitors. Altogether, this work will give unprecedented insight into the heterogeneity of HSC behavior and provide a platform to investigate the mechanisms that govern particular fate decisions.